Development of infection model for studying intracellular gene expression of Mycobacterium tuberculosis.

Mycobacterium tuberculosis complex owe their ability to cause infection because of their intracellular survival ability in professional phagocytic cells of human and the ability to enter into stage ofdormancy. The aim of this study was to develop an infection model that could be used to study M. tuberculosis and macrophage interactions at molecular level. Four infection models were examined namely opsonised M. bovis BCG / J774.2 macrophage cell line, non-opsonised M. bovis BCG / J774.2 macrophage cell line, opsonised M. tuberculosis / J774.2 macrophage cell line, and non-opsonised M. tuberculosis / J774.2 macrophage cell line infection models. A J774.2 macrophage cell line was synchronously infected with M. bovis (BCG strain) and M. tuberculosis (H37Rv), respectively at different multiplicity of infections (M.O.I). For opsonisation, the organisms were pre-incubated with human serum prior to infection. The infected cell lines were examined by light microscopy and electron microscopy with viable bacterial counts. Macrophage viability was assessed by trypan blue exclusion staining. The results showed higher significant level of infection of J774.2 macrophage cell line by opsonised M. bovis BCG (30 - 40%) compared to non-opsonised M. bovis BCG (< 0.1%) at an M.O.I of 50 (p < 0.05) with high macrophage viability. In contrast, there was no significant statistical difference (p > 0.05) in high infectivity (30 - 42%) with high macrophage viability achieved with using non-opsonised M. tuberculosis and opsonised M. tuberculosis, respectively, at an M.O.I of 10. In conclusion, opsonisation is not required for M. tuberculosis / J774.2 infection model in contrast to M. bovis BCG / J774.2 infection model where opsonisation is necessary to achieve high level of infection.

The Use of Rap-Pcr in Studying Mycobacterium Tuberculosis Intracellular Gene During Macrophage Infection

Mycobacterium tuberculosis is the second leading cause of death from infectious agent. This study sought to detect M. tuberculosis genes; which were specifically expressed; or upregulated during intracellular infection of J774 murine macrophages; as such genes may be potential targets for novel drug action. J774 murine macrophage cell line was infected with M. tuberculosis (H37Rv strain) at 10:1 multiplicity of infection (MOI). RNA was differentially extracted from M. tuberculosis infecting J774 macrophage cell line. The control in this case was RNA from extracellular broth grown bacteria. Approximately 50 ng of RNA from intracellular derived bacteria and extracellular derived bacteria (control) were subjected to random arbitrarily primed PCR (RAP-PCR) using 50 primer combinations. Eleven differential RAP-PCR products were observed. All RAP-PCR products were cloned into pCRr2.1 and sequenced in order to determine the identity of the products. Four of the eleven products were derived from macrophage genes and another 4 products were derived from the M. tuberculosis rRNA genes (three 23S and one 16S rRNA). The 3 remaining RAP-PCR products were found to be mycobacterial genes other than ribosomal genes. The three products were genes encoding enzyme involving in a shikimate pathway; a putative carboxyphosphonoenolpyruvate phosphonomutase and a serine protease with homology to HtrA. Of the 3 mycobacterial genes other than ribosomal genes detected; none were specifically expressed during intracellular infection but bacilli

Mycobacterial stationary phase induced by low oxygen tension: cell wall thickening and localization of the 16-kilodalton alpha-crystallin homolog.

Most cases of tuberculosis are due to reactivation of endogenous infection which may have lain quiescent or dormant for decades. How Mycobacterium tuberculosis survives for this length of time is unknown, but it is hypothesized that reduced oxygen tension may trigger the tubercle bacillus to enter a state of dormancy. Mycobacterium bovis BCG and M. tuberculosis H37Rv were cultured under aerobic, microaerobic, and anaerobic conditions. Their ultrastructural morphology was analyzed by transmission electron microscopy (TEM), and protein expression profiles were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). TEM revealed that the microaerobically and anaerobically cultured bacilli but not the aerobically cultured bacilli developed a strikingly thickened cell wall outer layer. The thickening was not observed in aerobically cultured stationary-phase bacilli or in anaerobically cultured Mycobacterium smegmatis. A highly expressed protein was detected by SDS-PAGE in microaerobic and anaerobic cultures and was identified as the 16-kDa small heat shock protein or alpha-crystallin homolog. Immunolocalization by colloidal gold immunoelectron microscopy identified three patterns of protein distribution in M. bovis BCG cultured under low oxygen tension. The 16-kDa protein was strongly associated with the cell envelope, fibrous peptidoglycan-like structures, and intracellular and peripheral clusters. These results suggest that tubercle bacilli may adapt to low-oxygen conditions by developing a thickened cell wall and that the 16-kDa protein may play a role in stabilizing cell structures during long-term survival, thus helping the bacilli survive the low oxygen tension in granulomas. As such, the cell wall thickening and the 16-kDa protein may be markers for the dormant state of M. tuberculosis.

Restriction fragment length polymorphisms in isolates of Aspergillus fumigatus probed with part of the intergenic spacer region from the ribosomal RNA gene complex of Aspergillus nidulans.

Differences in restriction fragment length polymorphisms (RFLPs) have been detected in isolates of Aspergillus fumigatus. Genomic DNA from 11 isolates was digested with EcoRI, separated by electrophoresis, Southern blotted and probed with DNA from the intergenic spacer or non-transcribed spacer region of the rRNA gene complex of Aspergillus nidulans. Three distinct RFLP patterns were detected which differed from the control patterns observed with A. nidulans, Aspergillus flavus and Aspergillus niger hybridized with the same probe. Furthermore, the differences in RFLP patterns in the A. fumigatus isolates were not detected when probed with DNA coding for the rRNA complex in Saccharomyces cerevisiae. These findings may be of use in the study of the epidemiology and pathogenesis of infections caused by A. fumigatus.

Improved methods for the preparation of high molecular weight DNA from large and small scale cultures of filamentous fungi.

Improved methods are described for the isolation of pure, high molecular weight DNA from small and large scale cultures of filamentous fungi. The methods depend on the extraction of DNA under conditions which prevent nuclease activity and contamination by carbohydrate. The small scale method depends on enzymatic digestion of the wall whereas the large scale method uses partial damage followed by autolysis. High yields of DNA are obtained by both methods and the DNA is suitable for restriction analysis. Southern Blotting, RFLP analysis, dot blotting and the production of gene libraries. The small scale method can be used for the simultaneous analysis of multiple cultures.

Bronchoalveolar lavage in an immunosuppressed rabbit model of invasive pulmonary aspergillosis.

A rabbit model of invasive aspergillosis has been used to investigate the pathogenesis of Aspergillus infection in the immunosuppressed host. The animals received hydrocortisone daily and a single dose of cyclophosphamide 2 days prior to intratracheal instillation of conidia from Aspergillus fumigatus. Bronchoalveolar lavage (BAL) was performed in 3 infected and 2 control saline treated animals sacrificed on days 1, 2, 4, 7 and 10 following inoculation. Infective load within the lung was quantified using an assay for chitin which is an important component of fungal cell walls (in particular the hyphal cell wall) and is not present in vertebrate tissue. The total BAL white cell count did not discriminate between infected and saline treated animals and Aspergillus was cultured from one lavage specimen only. Infected animals developed a marked neutrophil alveolitis by day 2 in contrast to a near total absence of neutrophils in the lavages of the control animals. Phagocytosis of conidia by alveolar macrophages was prominent but did not prevent progressive infection as confirmed by measurement of lung chitin. This pattern of cellular response within the alveolar airspace reflects the complex nature of the response to Aspergillus infection in the immunosuppressed host.

Invasive aspergillosis: clinical and pathological features of a new animal model.

A new animal model of invasive aspergillosis is described in which female New Zealand White rabbits were immunosuppressed with corticosteroids and cyclophosphamide and were given an intratracheal inoculation of 4 x 10(4) conidia of Aspergillus fumigatus. Thirteen of 15 animals survived during a 10-day-period of observation. Most had clinical signs of a respiratory infection (dyspnoea) and at autopsy there was macroscopic and microscopic evidence of invasive pulmonary aspergillosis. Six control animals (infected but not immunosuppressed) showed no such signs. The extent of hyphal invasion was assessed histologically and quantified by calculating the number of colony forming units (c.f.u.) g-1 of tissue: in the experimental group the mean c.f.u. value for the lungs was 1.25 x 10(3) compared to 73.3 c.f.u. g-1 of lung for the control group (P = 0.003). The infection was also quantified by a whole lung chitin assay: in the experimental group the mean chitin content (expressed as a glucosamine equivalent) was 3.05 micrograms g-1 lung tissue compared to a 0.53 micrograms g-1 lung tissue for the control group (P = 0.01). We conclude that this model of invasive aspergillosis in rabbits reflects closely the pathological features of the disease in man and that it may prove useful for studies of the pathogenesis and the treatment of invasive aspergillosis.